Intravascular hemolysis associated with Candidatus Mycoplasma hematoparvum in a non-splenectomized dog in the south region of Iran.

A 2-year-old male Pekingese dog was referred to Shiraz University’s Veterinary Teaching Hospital for anorexia and depression. The case had no history of surgery. Physical examination revealed no abnormalities except mild depression and fever. Small, coccoid, epicellular bacteria were detected on erythrocytes by microscopic examination of the Giemsa-stained blood smears. Abnormalities noted in the complete blood count included regenerative anemia characterized by a marked reticulocytosis. Examination of the plasma showed visual evidence of slight intra vascular hemolysis. In addition, Howell-Jolly bodies, nucleated RBCs, increased immature neutrophils and thrombocytosis were found in this case. The urine was strongly positive for bilirubin, and the urine sediment had abundant bilirubin crystals. For polymerase chain reaction (PCR) purpose, total DNA was extracted from blood sample collected from dog. PCR was positive and phylogenetic analysis of concatenated data showed our isolate clustered within Candidatus Mycoplasma hematoparvum group. Treatment was performed by oral ciprofloxacin and prednisolone. The clinical signs improved after three days. Two month follow-up showed no recurrence. In conclusion, hemoplasmosis should be considered as a differential diagnosis in dogs with hemolytic process and pyrexia. The PCR evaluation for hemoplasma DNA should be included in the investigation of such cases to enable the rapid detection of this infection, which may be more common than previously estimated. Besides, ciprofloxacin might have an effect on treatment of hemoplasma in dogs, however, conducting further case studies are necessary to recommend successful treatment.


Introduction
There are two species of hemotropic mycoplasma (also called hemoplasma) that infect dogs which are known so far: Mycoplasma hemocanis and Candidatus Mycoplasma hematoparvum (CMhp). 1,2 The main form of transmission is probably through blood sucking arthropods such as the tick Rhipicephalus sanguineus whose main geographical distribution is associated with the Mediterranean and sub-Mediterranean climates. 3 These extracellular parasites attach to the surface of canine erythrocytes, causing hemolytic anemia mostly through extravascular destruction of erythrocytes by the mononuclear phagocyte system. 2 Infection with these hemoplasmas generally only induces clinically significant anemia in splenectomized or immunocompromised dogs, although latent infections may cause subclinical anemia. 2,4 Candidatus M. hematoparvum was first described in association with anemia in a splenectomized dog undergoing chemotherapy for leukaemia. 5 Most non-splenectomized dogs infected with hemoplasma do not develop clinical evidence of disease and do not have sufficient number of organisms present in the blood to be recognized during routine blood film examinations. Besides, diagnosis of these pathogens by serological responses can be unspecific. Therefore, molecular techniques that are simpler, faster, less hazardous and usually more sensitive have been developed for hemoplasmas species detection. 2,6 The present case report describes the first report of hemolytic Mycoplasma infection in the south of Iran and hemolytic disorders in a non-splenectomized dog infected with CMhp.

Case Description
A 2-year-old male Pekingese dog was referred to Veterinary Teaching Hospital of Shiraz University for anorexia and depression. The case had no history of surgery. Body temperature was 40 ˚C, heart rate (130 beat per min), respiratory rate (20 breath per min) and body condition was normal. Physical examination revealed no abnormalities except mild depression.
Hematological examination was carried out using automatic cell counter (Exigo, Stockholm, Sweden) and blood smears were prepared for Giemsa staining.

Results
Small, coccoid, epicellular bacteria were detected on erythrocytes by microscopic examination of the Giemsastained blood smears (Fig. 1).
For polymerase chain reaction (PCR) purpose, total DNA was extracted from blood sample collected from dog using the DNeasy ® Blood and Tissue Kits (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Amplification of the 16S rDNA was performed using the lyophilized PCR micro tubes (Model Accupower PCR PreMix; BioNeer Co., Seoul, Korea) and oligonucleotides targeting 16S rDNA regions specific for HM (16S_ HEMO forw: GGCCCATATTCCT (AG) CGGGAAG; 16S_ HEMOrev: AC (AG) GGATTACTAGTGATTCCA). 7 The amplified products were sequenced directly using the capillary DNA analyzer (Model ABI 3730; Applied Biosystems, Foster City, USA). The 16S rDNA sequence obtained was compared to GenBank entries using the BLAST tool provided by National Center for Biotechnology Information (NCBI). 8 Different related Mycoplasma species were used for phylogenetic analysis. Multiple sequence alignments and construction of a phylogenetic tree were made with the neighbor-joining method using the software, MEGA (Version 4.0; Biodesign Institute, Tempe, USA). 9 PCR was positive by producing a specific fragment of ~1000 bp from DNA of the blood as shown in Figure 2.
Phylogenetic analysis of concatenated data showed our isolate clustered within the CMhp group (Fig. 3). Furthermore, comparative sequence analysis using the obtained 16S rDNA sequence (Accession number KC 762746) demonstrated the highest homology (more than 99.0%) to CMhp, previously described by Novacco et al. 10 (Accession numbers GQ129112, GQ129113: Italy) and Wengi et al. 3 (Accession number EF416569: Switzerland). The difference between Iranian sequence and these isolates was only a transition mutation of cytosine (C) to thymine (T) at position 902 based on the accession number KC762746. Also, comparison of an available sequence for CMhp (Accession number AY532390) originating from France with Iranian sequence showed the greatest difference (0.7%; 7 nucleotides).

Discussion
This is the first case of canine infection with CMhp in the southern region of Iran. This organism, named CMhp is smaller than M. hemocanis and does not form chains on the erythrocyte surface of dogs. 5 Candidatus M. hematoparvum is genetically more closely related to Candidatus M. hemominutum than to M. hemofelis, and was isolated from the blood of a splenectomized dog with hematopoietic neoplasia. 5 The infection has been confirmed by methods of molecular biology and specific PCR is the gold standard for detection of these infections. 12,13 Santos showed twenty (11.3%) out of 176 dogs living in rural areas were positive for hemoplasmas, whereas 6 of 104 (5.8%) dogs from urban areas harbored the organism. 13 Our case lived indoors in an urban area. Blood samples from 460 dogs living in the south of France showed 9.6% were infected with CMhp whereas only 3.3% were infected with M. hemocanis and 2.6% were infected with both organisms. 14 However, in Switzerland only 1.2% of dogs had positive real-time PCR results for the canine hemoplasmas. The prevalence in Europe is higher in the Mediterranean countries. It is postulated that the presence or absence of an appropriate vector for transmission of the organisms may explain these differences. 2 Clinical cases of canine hemoplasmosis have occasionally been reported, but co-factors such as splenectomy or immunosuppression seem to play a role in pathogenesis of the disease. Rare cases of acute disease have occurred in dogs with intact spleen in which no evidence for immunosuppression was found. 2 However, additional immunological assays (not routinely available) were necessary to better characterize the underlying immunosuppressive conditions. In this case, hemolysis and fever existed in non-splenectomized dog. So it could be postulated that in intravascular hemolysis, hemoplasmas must be considered in differential diagnosis as a suspected causative agent.
On the other hand, most non-splenectomized dogs infected with hemoplasmas do not have sufficient numbers of organisms present in the blood to be recognized during routine blood film examinations and due to unspecific serological examination, PCR could be recommended. In the present case, we demonstrated this hemoplasma species in a hemolytic dog with regenerative response. As mentioned above, a greater regenerative response occurs in hemolytic anemia than in other anemia because the iron and protein of the destroyed RBCs are readily available for erythropoiesis. The polychromasia, anisocytosis, Howell-Jolly bodies, and nucleated RBCs are consistent with regenerative anemia. 15 In spite of normal PCV (50.0%), ongoing slight intravascular hemolysis was confirmed with light pink to red discoloration of plasma and remarkable regenerative response. Hemoglobinuria was not detected in this case because hemoglobinemia occurred below the threshold for urinary excretion of hemoglobin (> 150 mg dL -1 ).
Recent research indicated that treatment of M. hemofelis infected cats with the fluoroquinolone may offer more effective long-term clearance of organisms than doxycycline. 2 Orally administered tetracyclines are reported to be effective in treating M. hemocanis infections but data are not available on the efficacy of treatment of CMhp infection in dogs. 2 We translated successful cat treatment for this case. The clinical signs improved within three days of initiating treatment and follow-up showed no regression.
This case was unique in several ways: first in describing CMhp infection using sequencing method in Iran. Since ticks and mites as important sources of Hemobartonella infection are frequent in this area, it seems this infection may be common in these dogs and all ticks should be removed from the environment; second, in associating hemoplasma infection with pyrexia and intravascular hemolysis in a non-splenectomized dog, and third, successful treatment.
In conclusion, hemoplasmosis should be considered as a differential diagnosis in dogs with hemolytic process and pyrexia. The PCR evaluation for hemoplasma DNA should be included in the investigation of such cases to enable the rapid detection of this infection, which may be more common than previously estimated. Besides, ciprofloxacin might have an effect on treatment of hemoplasma in dogs, however, conducting further case studies are necessary to recommend successful treatment.